The Fitness Costs and Regulatory Strategies of Cooperative Siderophore Production in Pseudomonas PublicDeposited

Descriptions

Cooperative behaviors in bacteria are increasingly appreciated for their relevance to microbial ecology and utility as model systems for social evolution. One example is the secretion of siderophores, a structurally diverse group of compounds that chelate extracellular iron. Siderophore production is considered cooperative because the benefits can be shared with neighboring cells, provided they have a suitable uptake system. As a cooperative behavior, evolutionary theory predicts siderophore production should be vulnerable to exploitation by free-loaders who reap the benefits without equal investment. Indeed, natural isolates often express receptors for siderophores they do not produce, indicating that social conflict is ecologically relevant. However, many questions remain unanswered regarding the influence of social conflict on the evolution of siderophore-producing populations. Specifically, the environmental conditions conducive to free-loading are not fully understood, obscuring efforts to distinguish whether siderophore-negative isolates evolved as social cheaters or are a result of non-social adaptations. It is also unknown how bacterial phenotypes are regulated to optimize fitness when competing with other siderophore producers. The results presented here provide insight into these two questions.
We first investigated the relationship between nutrient limitation and the fitness cost of pyoverdine (PVD) production, the primary siderophore of the medically relevant model organism Pseudomonas aeruginosa. Using metabolic modeling, we showed PVD production, although energetically costly, does not influence growth rate unless a building block of PVD (carbon or nitrogen) is limiting. When growth is limited by any other nutrient such that carbon and nitrogen are in relative excess, PVD production does not compete with the generation of cellular biomass and therefore does not reduce the growth rate. We confirmed these results experimentally with a continuous-culture approach. We showed that isogenic PVD-negative mutants act as free-loaders in co-culture with a PVD-producing wild-type when limited by carbon, but not when limited by phosphorus.
Second, we focused on the competitive strategies of soil bacterium Pseudomonas protegens Pf-5, a biocontrol strain remarkable for its ability to use dozens of siderophores it does not make. We found that while Pf-5 is able to regulate its receptors dynamically to reflect siderophore availability, it continues to secrete its own primary siderophore PVDPf-5, declining the opportunity to free-load. We demonstrated that this strategy is beneficial in co-culture with a competing PVD producer, P. aeruginosa PAO1. Although Pf-5 can use PAO1’s siderophore, Pf-5 must continue to produce its own to maintain a competitive advantage. We attribute this to an antagonistic effect of PVDPf-5 on the growth of PAO1, presumably through limiting access to iron. Our results demonstrate the benefits of excluding competitors exceed the incentives associated with a free-loader lifestyle for Pf-5.
Our findings are important not only for a fundamental understanding of microbial populations, but also for contexts highly relevant to humans, including human infections and agricultural applications. The principle of nutrient-dependent fitness costs has implications for the not only the stability of cooperation in the PVD model system, but also cooperation in general. Our work with Pf-5 emphasizes that complex strategies beyond free-loading are also relevant to siderophore social dynamics. Collectively, our findings contribute to a predictive understanding of how social dynamics influence the evolution and stability of siderophore production.